The Animal Welfare Act (AWA) and government agencies require, when available, the use of alternative methods which address the Reduction, Refinement and Replacement (3R's) of animal studies. The Local Lymph Node Assay (LLNA) is an alternative to the guinea pig sensitization test which is employed by the pharmaceutical, chemical, consumer product and cosmetic industries to identify and characterize substances with immunotoxic properties. The purpose of this proposal is to determine the feasibility of applying flow cytometry to enhance the LLNA. A major limitation of the LLNA in its current form is that it cannot differentiate between subclasses of sensitizing agents (i.e., contact vs. respiratory allergens). Furthermore, the assay uses radioactivity and large numbers of animals. Flow cytometry-based endpoints will enhance the sensitivity and discriminating power of the LLNA, while (1) complying with the AWA by directly addressing the 3R's; (2) increasing the quality and quantity of data generated when compared to existing methods (guinea pig test); and (3) substantially reducing the cost of analysis by avoiding the use of radioactivity. The development of this enhanced assay will satisfy a demand in the industry and test the feasibility of our long term plan to apply flow cytometry to existing assays and to develop new toxicity tests. PROPOSED COMMERCIAL APPLICATIONS: MB Research will offer the LLNA using flow cytometry to the pharmaceutical, biotech, cosmetic, chemical and consumer products industries. Included with this proposal are letters of support from several major companies (Merck, Johnson & Johnson Advanced Care Products, ISP, and Purdue Pharma) demonstrating interest in an enhanced LLNA. A survey of existing clients indicates a great demand for the LLNA and other flow cytometric-based assays (see Relationship to Future Research and Development). Flow cytometry is a powerful technology that is underutilized in toxicology and pharmacology testing which, in addition to improving the marketability of the LLNA, can be applied to develop other commercially viable tests.